Download Are cerebral creatine deficiency syndromes on the

REVIEW
Special Focus
Are cerebral creatine deficiency syndromes
on the radar screen?
Lígia S Almeida,
Efraim H Rosenberg,
Nanda M Verhoeven,
Cornelis Jakobs
& Gajja S Salomons†
†Author
for correspondence
VU University Medical
Center, Department of
Clinical Chemistry,
Metabolic Unit,
De Boelelaan 1117,
1081 HV Amsterdam,
The Netherlands
Tel.: +31 204 442 914;
Fax: +31 204 440 305;
[email protected]
Keywords: AGAT, cerebral
creatine deficiency syndromes,
creatine, GAMT, GATM,
guanidino compounds,
mental retardation, metabolic
disorders, neuromodulator,
SLC6A8, solute carrier family
6 member 8
Cerebral creatine deficiency syndromes (CCDS) are responsible for a considerable
proportion of the population affected with mental retardation. CCDS are caused by either
an inborn error of the proteins involved in creatine biosynthesis or in the creatine transporter.
Besides mental retardation, the clinical characteristics of CCDS are speech and language
delay, epilepsy and features of autism. CCDS can be diagnosed by proton magnetic
resonance spectroscopy of the brain and/or by biochemical and molecular analysis.
Treatment of the defects in creatine biosynthesis has yielded favorable outcomes, while
treatments for creatine transporter deficiency are still under investigation at this time. The
relatively large contribution of the CCDS to the monogenic causes of mental retardation
emphasizes the importance of including CCDS in the differential diagnosis of mental
retardation of unknown etiology. Pathophysiology is not yet unravelled, although it is known
that creatine plays an important role in energy storage and transmission. Moreover, in vitro
data indicate that creatine acts as a neuromodulator in the brain.
In the last decade, a novel group of inborn errors
of proteins involved in creatine biosynthesis and
its transporter has been identified. The high prevalence of these defects within the mentally
retarded population and the promising treatment
possibilities argue for the inclusion of cerebral
creatine deficiency syndromes (CCDS) in the differential diagnosis of mental retardation of
unknown etiology. CCDS arise by mutations in
either one of the autosomal genes AGAT and
GAMT, which encode the two enzymes
(arginine:glycine amidinotransferase and guanidinoacetate methyltransferase, respectively) [1,2]
necessary for creatine biosynthesis, or by mutations in the X chromosomal creatine transporter
gene (solute carrier family 6 member 8
[SLC6A8]) [3]. Until recently, this group of syndromes has been termed creatine deficiency syndromes (CDS). However, in bodily fluids, no
creatine deficiency exists in creatine transporterdeficient patients, thus, this term may be misleading. We therefore prefer to use the term CCDS,
which also correlates better to the main clinical
hallmarks that are related to CNS involvement.
In the general population, the incidence of
mental retardation is estimated to be approximately 1% (profound, severe and moderate cases)
to 3% if mild cases are included (IQ 50–70).
Within the mentally retarded population, the frequency of X-linked mental retardation (XLMR) is
estimated at 5–12% [4]. This number, and the fact
that a large group of XLMR genes are already
known [5], indicates that each monogenic cause of
XLMR only accounts for a small percentage of
10.2217/14796708.1.5.637 © 2006 Future Medicine Ltd ISSN 1479-6708
mental retardation in males [6,7]. For the fragile X
mental retardation gene (FMR1), the Aristalessrelated homeobox gene (ARX) and, to a lesser
extent, for the creatine transporter gene (SLC6A8),
this seems to be different, as they account for a
larger proportion of the XLMR subgroup [7].
The prevalence of SLC6A8 deficiency has
been studied in three different patient groups. In
the first panel, consisting of males with a strong
predilection to having XLMR, a prevalence of
2.1% (six out of 288) was found. Considering
the estimation that only 10% of the patients
with mental retardation are affected with an
X-linked defect, the prevalence of 2.1% in
XLMR would translate to approximately 0.2%
in the general mental retardation population of
unknown etiology [8]. However, the prevalence
in the two panels with mental retardation (four
out of 478 = 0.8%, [9]) and global development
delay (two out of 92 = 2.2%, [10]) was somewhat
higher than predicted. This latter group was
investigated by proton magnetic resonance spectroscopy (MRS), whereas the other groups were
studied by DNA sequence analysis.
The prevalence of AGAT and GAMT deficiencies are not expected to be high, since they
are autosomal recessive disorders. Indeed, only
29 GAMT- and five AGAT-deficient patients
have been reported [11–15]. However, awareness
of GAMT deficiency may be of utmost importance in Mediterranean countries, because a high
carrier rate of a pathogenic GAMT mutation
exists [16,17], and most of the GAMT-deficient
patients are from this region.
Future Neurol. (2006) 1(5), 637–649
637
REVIEW – Almeida, Rosenberg, Verhoeven, Jakobs & Salomons
The relatively high prevalence of CCDS highlights the importance of CCDS inclusion in the
differential diagnosis of mental retardation of
unknown etiology.
Inborn errors of proteins involved in
creatine biosynthesis & its transporter
Arginine:glycine
amidinotransferase deficiency
The initial and rate-limiting step in creatine biosynthesis is catalyzed by the AGAT enzyme,
which forms guanidinoacetic acid, the precursor
of creatine (Figure 1). In 2000, the first siblings
with a defect in this enzyme were recognized [18].
These sisters (4- and 6-years old) presented with
mental retardation and severe language delay.
Routine blood and urine analyses were normal
and further investigations did not suggest a
Figure 1. Creatine biosynthesis.
Arginine
Guanidinoacetate
methyltransferase deficiency
Glycine
AGAT
Ornithine
Guanidinoacetic acid
SAM
GAMT
SAH
Creatine
Phosphocreatine
Creatine
CK
SLC6A8
ADP ATP
H2O
Creatine
Creatinine
Creatine biosynthesis involves a two-step reaction: the first is catalyzed by
arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) and the second by
guanidinoacetate methyltransferase (GAMT, EC 2.1.1.2). Creatine is
transported via the bloodstream and taken up by tissues with high CK activity,
such as muscle and brain, via a creatine transporter (SLC6A8). CK catalyzes the
phosphorylation and dephosphorylation of creatine and phosphocreatine,
respectively, thus providing a high-energy phosphate buffering system during
ATP release and use.
ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; CK: Creatine kinase.
638
neurometabolic disorder. Magnetic resonance
imaging (MRI) of the brain was normal, but
proton MRS revealed the absence of the creatine–phosphocreatine signal, suggesting a defect
in the proteins involved in creatine biosynthesis
or in the creatine transporter. In plasma, concentrations of creatine and guanidinoacetic acid
were within normal values, which ruled out a
deficiency of GAMT. The patients were reinvestigated in 2001, and analysis of urine revealed
consistently decreased levels of guanidinoacetic
acid [19]. The diagnosis of AGAT deficiency was
confirmed by impaired AGAT activity in cultured cells and the detection of a pathogenic
mutation in exon 3 of the AGAT gene [19].
Presently, five AGAT-deficient patients are
known from two unrelated families, in whom
reduced levels of GAA in body fluids have been
detected [14,15]. The general clinical and
biochemical findings are listed in Table 1.
The second step in creatine biosynthesis is mediated by the GAMT enzyme (Figure 1). In 1994, the
first GAMT-deficient patient, a 22-month-old
male was identified by Stöckler and collaborators [20,21]. The onset of symptoms started at
5 months with developmental arrest. The patient
was hypotonic, unable to sit or roll over, showed
uncoordinated swallowing and developed a severe
extrapyramidal disorder. He had no organomegaly
and his head circumference, hearing and vision
appeared normal. Electrocardiogram and cardiac
ultrasound examinations were normal; electroencephalogram showed low background activity
and multifocal spikes. Nonspecific biochemical
elevations were reported due to decreased creatinine levels. Magnetic resonance studies were performed for the first time at the age of 12 months:
MRI revealed bilateral abnormalities in the globus
pallidus. Proton MRS showed a spectrum lacking
a creatine signal and an elevated guanidinoacetic
acid peak. Treatment with arginine, the substrate
of the AGAT enzyme, did not result in restoration
of brain creatine. Moreover, guanidinoacetic acid
in the brain, measured by proton MRS, remained
increased, indicating that the defect was caused by
a block in GAMT activity [20]. Indeed, in 1996,
impaired GAMT activity in cultured cells and
pathogenic mutations in the GAMT gene were
identified [21]. Furthermore, a GAMT knockout
mouse model has been developed, mimicking the
biochemical characteristics of GAMT deficiency
in humans [22–24].
Future Neurol. (2006) 1(5)
Are cerebral creatine deficiency syndromes on the radar screen? – REVIEW
Table 1. Overview of the patients described to date.
Cerebral creatine
deficiency
AGAT
(n = 5)§
GAMT
(n = 29)¶
SLC6A8
(n = 24)#
Age at diagnosis
0–5
0–26
2–66 years
Developmental delay
5/5
27/29
24/24
Speech and language delay
5/5
21/29
24/24
Mental retardation
5/5
27/29
24/24
Hypotonia
2/5
27/29
11/16
Behavior disorder
1/5
21/29
14/19
Movement disorder
NR
15/29
10/19
Seizures
NR‡
25/29
16/24
Mild phenotype*
NA
3/29
NA
Intermediate phenotype*
NA
12/29
NA
Severe phenotype*
NA
12/29
NA
H-MRS of the brain:
absence/reduction Cr
5/5
27/29
12/12
Clinical traits:
Biochemical findings:
Urinary creatine:creatinine
Increase in 17/17
Urinary guanidinoacetic acid
Decrease
in 5/5
Increase in
29/29
Increase in 2/4
Plasmatic guanidinoacetic
acid
Decrease
in 5/5
Increase in
28/28
Increase in 1/2
Plasmatic creatine
Decrease
in 5/5
Decrease in
28/28
NR
Treatment available
Yes
Yes
?
*Based on [11].
‡Febrile
seizures were reported in one of the first patients to be described [18].
§ [14,15,19]; ¶ [11,12,13]; #[9,53,66,67].
AGAT: Arginine–Glycine amidinotransferase; GAMT: Guanidinoacetate
methyltransferase; NA: Not applicable; NR: Not reported.
To date, 29 patients have been described, varying from neonate to 29 years of age [11–13].
Owing to heterogeneous clinical presentation,
GAMT-deficient patients can be classified as
having a mild, moderate or severe phenotype,
based on the severity grade of the main clinical
characteristics (i.e., mental retardation, epilepsy
and movement disorder). An overview of the
clinical characteristics reported so far is
presented in Table 1.
Creatine transporter deficiency
The creatine transporter, encoded by the SLC6A8
gene, is essential for creatine uptake into cells. In
2001, the first patient with SLC6A8 deficiency
was described. The patient presented with mild
developmental delay at the age of 7 months in
combination with central hypotonia. Prenatal and
perinatal histories were unremarkable [25]. There
was a history of learning disabilities and mental
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retardation in the family, compatible with an
X-linked disorder. At 2 years of age the patient
was admitted to hospital owing to a partial status
epilepticus. Examination at 6 years of age showed
a severe delay in speech and language development. A progressive increase of the head circumference (75th percentile to 95th percentile)
prompted MRI and proton MRS. Proton MRS
highlighted an almost complete loss of the creatine and phosphocreatine signal, similar to that
observed in patients with AGAT and GAMT deficiency [26]. Consequently, oral creatine supplementation was commenced. However, after
4 months no restoration of cerebral creatine concentration was observed, which was in line with
the lack of clinical improvement. Therefore, oral
creatine supplementation was discontinued.
This, and the biochemical findings, ruled out
a creatine biosynthesis defect. Moreover, high
urinary creatine concentrations suggested a
defect in cellular creatine transport. The inheritance pattern suggestive for X-linked disease and
the fact that the gene encoding the creatine
transporter, SLC6A8, is mapped to Xq28 [27],
strengthened this hypothesis. This transporter is
a member of the Na+/Cl--dependent neurotransmitter transporter family (Figure 2). Indeed,
sequence analysis of SLC6A8 revealed a
hemizygous nonsense mutation. Furthermore,
impaired creatine uptake in cultured fibroblasts
was demonstrated [28]. The carrier status for this
mutation was confirmed in the female relatives.
So far, more than 34 patients have been
reported with SLC6A8 deficiency. The clinical
characteristics of 24 male patients are listed in
Table 1. In females, a very heterogeneous clinical
phenotype is expected due to skewed X-inactivation, varying from learning disabilities to
mental retardation.
How to diagnose cerebral creatine
deficiency syndromes?
A marked reduction of cerebral creatine measured
by proton MRS is highly indicative of a primary
creatine deficiency syndrome. Workup by metabolite, molecular and functional studies is required
to identify the underlying defect (i.e., AGAT,
GAMT or SLC6A8 deficiency). Additionally, in
families where individuals have previously been
diagnosed with CCDS, prenatal diagnosis can be
performed at the molecular level in chorion villus
sampling or amniotic cells [12,15]. In case of
GAMT deficiency, prenatal diagnosis can also be
performed from the amniotic fluid at the metabolite level [12]. At present, the primary choice for
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Published single amino acid mutations are depicted as blue squares. Cys144 is marked as a green hexagon [62]. Possible N-glycosylation substrates N192, N197 and N548 are depicted as
black hexagons. The putative Leu-zipper (L286, L293, L300 and I307) is marked with purple upwards arrows. Putative cyclic adenosine monophosphate phosphokinase phosphorylation
sites T56 and S256 are shown as orange downwards arrows. The figure was plotted with TOPO2 (Johns SJ, TOPO2, Transmembrane protein display software [101]).
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Figure 2. Schematic representation of SLC6A8 with the putative 12 transmembrane domain structure.
REVIEW – Almeida, Rosenberg, Verhoeven, Jakobs & Salomons
Future Neurol. (2006) 1(5)
Are cerebral creatine deficiency syndromes on the radar screen? – REVIEW
screening of CCDS in most institutes is measuring creatine, creatine:creatinine ratio and guanidinoacetic acid in body fluids or performing
proton MRS of the brain. In contrast with
metabolite analysis, proton MRS is expensive,
not widely available and sedation is usually
required.
Figure 3. Schematic representation
of the AGAT gene and its
reported mutations.
5´UTR
Ex1
Ex2
Metabolite analysis
Molecular diagnosis
Molecular diagnosis is usually performed by
direct sequencing of the open reading frame of
the respective gene or by mutation scanning
using DHPLC [36] followed by DNA sequence
analysis in case of aberrant heteroduplexes.
• The AGAT gene (Gene ID 2628, official
nomenclature: GATM) has been mapped to
chromosome 15q15.3, is 16.8 Kb in size and
contains nine exons, which encode a protein
of 424 amino acids. There are two mutations
described (Figure 3);
• The GAMT gene (Gene ID 2593) has been
mapped to chromosome 19p13.3, its size is
4.46 Kb and it contains six exons, which
encode a protein of 237 amino acids. There
are 15 pathogenic mutations described
throughout the gene (Figure 4);
• The creatine transporter gene – SLC6A8 (Gene
ID 6535) has been mapped to Xq28 [37–39].
The SLC6A8 gene spans 8.4 Kb consisting of
13 exons, which encode a protein of 635
amino acids. To date, 20 mutations have been
described throughout SLC6A8 (Figure 5).
Trp 149X
Ex3
c.484 + 1G>T
Ex4
Ex5
Ex6
Ex7
Ex8
Ex9
3´UTR
Various methods to measure creatine and guanidinoacetic acid in bodily fluids are available [29]. In
diagnostic settings, the techniques generally used
are gas chromatography-mass spectrometry [30,31],
high-performance liquid chromatography (HPLC)
[32] and tandem mass spectrometry [33–35].
Increased guanidinoacetic acid levels in bodily fluids are pathognomonic for GAMT deficiency,
whereas reduced levels are found in AGAT deficiency. Creatine is usually reduced in the bodily
fluids of patients with a biosynthesis defect, whereas
an increased urinary creatine:creatinine ratio is
found in males with SLC6A8 deficiency [30]. In the
majority of females with a heterozygous SLC6A8
mutation, this ratio is not informative.
200 nucleotides
3000 nucleotides
Exons (numbered boxes) and introns (black lines)
are drawn to scale. To date, only two mutations
have been reported (c.446G>A, p.Trp149X [19];
c.484 +1G>T, IVS3+1G>T [15]).
substrates [19,21,40,41]. In 2003, two methods
using stable isotope labeled substrates were
developed for both AGAT and GAMT [42,43].
The use of stable isotopes increases the sensitivity
of the methods and reduces the amount of
biological material required.
To study SLC6A8 deficiency, the creatine
transporter function can be tested by a functional
assay in fibroblasts [28].
Diagnostic pitfalls
Enzyme analysis
Several assays have been reported to assess both
AGAT and GAMT activity [29]. The first methods to be reported used radioactive-labeled
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In the last decade, only two families with AGAT
deficiency were reported, in comparison with
22 families affected with GAMT deficiency.
AGAT-deficient patients could remain unnoticed
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REVIEW – Almeida, Rosenberg, Verhoeven, Jakobs & Salomons
5´UTR
Figure 4. Schematic representation
of the GAMT gene and its
reported mutations.
Trp20Ser
Met50Leu
Hiss51Pro
Ala54Pro
Ex1
Ala22GlyfsX19
Ex2
g.1637-1787del
Arg105GlyfsX13
c.327G>A
Ex3
Asp135Asn
Cys169Tyr
Trp174X
Ex4
Ex5
Gly164AlafsX13
Gly164GlyfsX26
Glu176GlufsX13
c.571-3C>G
Leu197Pro
3´UTR
Ex6
100 nucleotides
20000 nucleotides
Exons (numbered boxes) and introns (black lines)
are drawn to scale. Splice-site and frameshift
mutations are indicated on the right side; missense
mutations are indicated on the left side; mutations
are indicated in terms of protein. References for the
mutations: c.59G > C, p.Trp20Ser; c.152A > C,
p.His51Pro; c.160G > C, Ala54Pro; c.64dupG,
p.Ala22fsX19; c.299_311dupGGGACTGGGCCCC,
p.Arg105GlyfsX13; c.491delG, p.Gly164fsX13;
c.521G > A, p.Trp174X; c.526dupG,
p.Glu176GlufsX13; c.327G > A, IVS2+1G > A;
g.1637_1787del [11]; c.506G > A,
p.Cys169Tyr [16]; c.148A > C, p.Met50Leu [12];
c.491dupG, p.Gly164fsX26; c.571–3G > C,
IVS5–3G > C [63]; c.590T > C, p.Leu197Pro [64].
in metabolic screening due to techniques that are
neither sensitive or specific enough to detect
decreased metabolite levels. Furthermore, intake
of nutrition containing high concentrations of
creatine (i.e., meat and fish) may hamper
diagnosis of AGAT deficiency [44].
642
In approximately 10% of the males with mental
retardation in whom elevated urinary creatine:creatinine ratio is found, no SLC6A8 mutation is
detected. They are likely to represent false-positive
outcomes. Elevated urinary creatine:creatinine
ratio may also be the secondary result of other
(neuro)muscular disorders, such as Duchenne
muscular dystrophy and Becker muscular dystrophy [45]. In rare cases, promoter mutations or deep
intronic mutations may have been missed.
Although three female SLC6A8-deficient
patients were found with an elevated urinary creatine:creatinine ratio, this is not the rule for the
majority of heterozygous females [29]. Proton
MRS of the brain in females may be restricted by
difficulties in detecting minimal reductions in
brain creatine and sedation is often required.
Currently, in females, molecular screening is
probably the most sensitive method for the
detection of SLC6A8 deficiency, and thus urinary creatine:creatinine ratio is not the primary
choice. In molecular diagnostic screening, care
should be taken, since a paralogous gene,
SLC6A10, which is mapped at chromosome
16p11, is highly identical to SLC6A8 [46].
Regarding DNA sequence analysis, it should be
noted that, in rare cases, unique sequence variations are detected, both in coding and noncoding
regions in the SLC6A8, GAMT or AGAT gene,
which were not encountered in controls [8,9,16].
Thus, it is important to evaluate the nature of the
variants carefully, before classifying them as
pathogenic mutations or polymorphisms.
Treatment of creatine biosynthesis
defects & creatine transporter defects
Treatment approaches of CCDS aim to restore
creatine in the brain by creatine supplementation,
with success for the biosynthesis defects [20,47,48].
For AGAT deficiency, the highest success rate is
expected because there is no accumulation of
substrates. In fact, if started in a presymptomatic
phase, creatine supplementation in AGAT and
GAMT-deficient patients leads to an impressive
restoration of cerebral creatine levels, and a favorable clinical response. In an AGAT-deficient
infant, treatment initiated at 4 months of age
showed normal development by 18 months of
age, and the restoration of creatine levels in bodily fluids and the brain were almost complete [49].
Similar success was observed with a GAMT-deficient patient in whom presymptomatic treatment
was initiated at 22 days of age [13]. This suggests
that creatine supplementation in early life
prevents the neurological sequelae.
Future Neurol. (2006) 1(5)
Are cerebral creatine deficiency syndromes on the radar screen? – REVIEW
Figure 5. Schematic representation
of the SLC6A8 gene and its
reported mutations.
5´UTR
Ex1
Gly87Arg
SLC6A8 del
Ex2
c.263-2A>G
Phe107del
Currently, the most extensive experience has
been obtained with the treatment of GAMTdeficient patients. The aim of the treatment is to
increase the creatine and decrease the guanidinoacetic acid in brain. The severe phenotype
observed in some cases in GAMT-deficient
patients might be due to both the lack of creatine,
but also due to guanidinoacetic acid accumulation, since guanidino compounds are known for
their neurotoxic and epileptogenic effects [50]. In
order to lower guanidinoacetic acid levels, AGAT
activity needs to be reduced. This can be partly
achieved by two different strategies:
• Substrate deprivation of AGAT by dietary
restriction of arginine intake;
• With supplementation of high concentrations
of ornithine and creatine.
Ex3
Ex4
Lys293fsX3
Ex5
Asn336del
c.1016+2T>C
Ile347del
Ex6
Arg391Trp
Ex8
Ex7
Ex9
Ex10
Ex11
Ex12
Tyr262X
Tyr317X
Cys337T rp
Gly381Arg/splice
Pro390Leu
Phe408del
c.1495+5G>C
Arg514X
Pro544Leu
Pro554Leu
Ex13
c.1255-?_?del
3´UTR
Creatine: a novel neuromodulator?
250 nucleotides
250 nucleotides
Exons (numbered boxes) and introns (black lines)
are drawn to scale. References for the mutations:
c.259G > A, p.Gly87Arg; c.319_321delCTT,
p.Phe107del; c.950_951insA, p.Tyr317X;
c.1011C > G, p.Cys337Trp; c.1169C > T,
p.Pro390Leu; c.1661C > T, p.Pro554Leu [8];
c.263–2A > G, IVS2–2A > G [65];
c.319_321delCTT, p.Phe107del; c.786C > G,
p.Tyr262X; c.1221_1223delCTT, p.Phe408del;
c.1141G > C, p.Gly381Arg/splice; c.1540C > T,
p.Arg514X [66]; c.878_879delTC, p.Lys293fsX3;
c.1221_1223delCTT, p.Phe408del; [53];
c.1006_1008delAAC, p.Asn336del;
c.1016+2T > C, IVS6+2T > C; c.1059_1061delCTT,
p.Ile347del; c.1171C > T, p.Arg391Trp [9];
c.1255?_?del; SLC6A8 del [67].
www.futuremedicine.com
With this therapeutic approach, levels of guanidinoacetic acid are lowered by 50% in bodily fluids [51]. This led to an additional clinical
improvement as compared with treatment with
creatine alone.
No successful treatment has currently been
reported for SLC6A8 deficiency. Attempts with
creatine supplementation in males showed no
marked improvement [25,28,52,53]. Current trials
are aiming at stimulation of creatine biosynthesis
in the brain by supplementation with high doses
of arginine and glycine, combined with high
doses of creatine (Mancini GM, van der Knaap MS, Salomons
GS, Unpublished Data). However, despite the expression of biosynthesis proteins in the brain, the spatial distribution of AGAT, GAMT and SLC6A8
may pose problems for this approach [54,55].
Whilst the function of creatine in energy metabolism has been addressed extensively, only a limited
number of studies have focused on its role in the
brain. Creatine synthesis has been observed in the
CNS [56]. In situ hybridization studies also found
AGAT and GAMT expression in almost all CNS
cell types, in addition to its expression at the
blood–brain barrier level, whereas SLC6A8
mRNA was only found in neurons, oligodendrocytes and brain capillary endothelial cells [54,55,57].
These data support the recent postulations that
the brain is able to synthesize creatine.
Creatine seems to be essential for brain function, as patients suffering from a CCDS have
mental retardation, speech and language disorders, autistic features and may have extrapyramidal movement disorders and epileptic seizures
(see above). Additionally, several guanidino
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REVIEW – Almeida, Rosenberg, Verhoeven, Jakobs & Salomons
compounds are neurotoxic, presumably by altering the functioning of inhibitory and excitatory
amino-acid receptors [58]. Considering the effects
of creatine on central neurotransmission processes, it has been shown that guanidino
compounds, including creatine, may affect γ-aminobutyric acid (GABA)-ergic neurotransmission
as (partial) agonists for GABAA receptors [58–60].
Indeed, it was shown that creatine is released
from central neurons in a manner similar to that
of classical neurotransmitters, specifically, involving an exocytotic release mechanism [61]. In view
of this in vitro data, it was hypothesized that creatine represents a cotransmitter in the brain that
modulates the functioning of postsynaptic receptors for neurotransmitters, such as GABA
(Figure 6) [61].
Conclusions
In the last decade, a novel group of inborn errors
of metabolism has been identified: the cerebral
creatine deficiency syndromes. The importance
of considering CCDS in the differential diagnosis of mental retardation is emphasized by the
relative high frequency of SLC6A8 deficiency,
and the promising results of treatment of
GAMT- and AGAT-deficient patients. The diagnostic tests to detect these syndromes are readily
Figure 6. Proposed model of action for cerebral creatine.
Pre-synaptic neuron
Arg +Gly
AGAT
GAA
GAMT
Creatine
Creatine
Ca2+
+ Na+
Glial cell
Ca2+
Ca2+
Ca2+
+ Na+
Creatine
SLC6A8
GABAA rec
Cl-/GABA-ergic inhibition
Post-synaptic neuron
Creatine is synthesized in neurons, by the enzymes AGAT and GAMT. Upon synthesis creatine is released in a
Ca2+ dependent (exocytotic/vesicular) manner into the synaptic cleft interacting with the GABAA receptors of
the postsynaptic neuron. Creatine may subsequently be taken up from the cleft via the creatine transporter
into the neurons or glial cells [61].
AGAT: Arginine:glycine amidinotransferase; GAA: guanidinoacetic acid; GABA: γ-amino butyric acid;
GAMT: Guanidinoacetate methyltransferase.
644
Future Neurol. (2006) 1(5)
Are cerebral creatine deficiency syndromes on the radar screen? – REVIEW
available (e.g., metabolite and molecular analysis and, to a lesser extent, proton MRS), which
makes this feasible for any institution that has
access to at least one of these techniques.
Moreover, proton MRS/MRI in particular enables the detection of multiple diseases. The recent
findings that creatine acts as a neuromodulator
opens up new research areas, which may be worthwhile for the elucidation of the pathophysiology
of CCDS.
Future perspectives
Within the next 5–10 years in the Western
world, every mentally retarded patient
(male/female) will be screened for CCDS. This
could be achieved primarily by proton MRS,
which will certainly become more widely available, with the advantage of disclosing additional
diseases. For the screening of CCDS, proton
MRS is adequate, as a marked reduction or
absence of the creatine signal is diagnostic for
primary creatine deficiency. Currently, molecular
analysis is performed by direct DNA sequencing.
However, current developments in the field of
microarrays allow the analysis of multiple genes,
or even full genomes, in a single assay, which
opens up a new world in both research and clinical applications. This technology may lead to
more insights into the pathophysiology. The
development of sequencing chips (i.e., resequencing arrays) also discloses new perspectives
in the diagnosis and screening of CCDS,
although its sensitivity is not yet acceptable for
clinical diagnostics.
Treatment of SLC6A8 deficiency is one of the
big challenges. Clinical improvement has been
observed in the patients with biosynthesis defects
upon treatment, with almost complete restoration of creatine in the brain. This proves that
cerebral creatine restoration is essential. When a
vehicle for creatine uptake in the brain is found,
treatment should also be successful for SLC6A8
deficiency. Elucidation of the creatine biosynthesis pathway in the brain, in addition to the clarification of the function of creatine in the brain,
may increase the success rate of treatment.
Tandem mass spectrometry has the potential
for simultaneous multidisease screening and has
recently been applied to neonatal screening programs. In case measurement of guanidinoacetic
acid in dried blood spots proves to be specific and
sensitive enough for detection/exclusion of
GAMT deficiency, this disorder will be included
in neonatal screening [32,33,13]. AGAT and
SLC6A8 deficiencies are not yet eligible for neonatal screening since creatine and creatinine do
not seem to be informative in the neonatal period.
Acknowledgements
Ligia S Almeida and Efraim H Rosenberg contributed
equally to this work. The work of Ligia S Almeida, Efraim
H Rosenberg and Gajja S Salomons was supported by the
Dutch Society for Scientific Research (ZonMW/NWO),
VIDI grant number 917.56.349.
Executive summary
Inborn errors of proteins involved in creatine biosynthesis & its transporter
• Three novel disorders in creatine metabolism have been identified. Two autosomal recessive creatine biosynthesis defects (AGAT
and GAMT deficiency) and one X-linked creatine transporter defect (SLC6A8 deficiency). This group is defined as cerebral creatine
deficiency syndromes (CCDS).
• The common hallmarks of CCDS are the absence, or marked reduction of the creatine signal in the proton magnetic resonance
spectroscopy (MRS) of the brain, mental retardation and speech and language delay (Table 1).
• Worldwide, 29 patients with GAMT deficiency, five with AGAT deficiency and over 34 with SLC6A8 deficiency have been reported.
High prevalence of cerebral creatine deficiency syndromes in the mental retardation population
• The prevalence of SLC6A8 deficiency in patients with mental retardation is relatively high (~1%) compared with most monogenic
causes of mental retardation.
• The prevalence of the autosomal recessive disorders AGAT- and GAMT-deficiency, is not high, however, a high carrier rate of a
GAMT (founder) mutation exists in Mediterranean countries.
• The relatively high prevalence of CCDS highlights the importance of including them in the differential diagnosis of mental
retardation of unknown etiology.
How to diagnose cerebral creatine deficiency syndromes?
• CCDS may be found either by proton MRS, metabolite screening and/or molecular investigations.
• Biochemically, AGAT-deficiency is characterized by low creatine and guanidinoacetic acid levels in bodily fluids, whereas
GAMT-deficiency is characterized by elevated levels of guanidinoacetic acid in bodily fluids.
• Elevated urinary creatine:creatinine ratios are found in males affected with SLC6A8 deficiency.
• In females, the urinary creatine:creatinine ratio is not usually informative in this X-linked disorder, thus mutation analysis of
SLC6A8 is currently the primary choice.
• Prenatal diagnosis of CCDS is possible in families with affected individuals.
www.futuremedicine.com
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REVIEW – Almeida, Rosenberg, Verhoeven, Jakobs & Salomons
Executive summary
Diagnostic pitfalls
• AGAT deficiency is likely under-diagnosed because the biochemical tests may not be sensitive or specific enough to detect
decreased metabolite levels.
• In approximately 10% of males with mental retardation in whom elevated urinary creatine:creatinine is found, no SLC6A8
mutation is detected.
• False-negative biochemical findings (i.e., normal urinary creatine:creatinine ratio) are found frequently in females with
heterozygous SLC6A8 mutations.
• In molecular diagnostic screening, proper criteria should be used to classify novel sequence variants in the CCDS genes as
pathogenic mutations.
Treatment of creatine biosynthesis defects & creatine transporter defect
• Treatment approaches of CCDS aim to restore cerebral creatine levels.
• The successful treatment of creatine biosynthesis defects consists primarily of daily creatine supplementation, which preferably
should be initiated early in life. In addition, in GAMT deficiency, guanidinoacetic acid levels can be reduced by arginine restriction
and ornithine supplementation.
• Current trials for the treatment of SLC6A8 deficiency aim at the stimulation of cerebral creatine biosynthesis by supplementation
with high dosage of the substrates of the AGAT enzyme (i.e., arginine and glycine).
Creatine: a novel neuromodulator?
• Cerebral creatine biosynthesis has been proven by the demonstration of AGAT and GAMT mRNA expression in the brain.
• In vitro data indicate that creatine represents a novel cotransmitter in the brain.
Conclusions
• Cerebral creatine deficiency syndromes are, in part, treatable disorders.
• Diagnostic tests for the detection of cerebral creatine deficiency syndromes are widely available.
• Cerebral creatine deficiency syndromes should be included in the differential diagnosis of mental retardation, owing to their
high frequency.
Future perspectives
•
•
•
•
In the Western world, every mentally retarded patient (male/female) will be screened for CCDS, primarily by proton MRS.
Novel techniques will increase the number of patients detected with CCDS.
By identifying a vehicle for creatine uptake in the brain, treatment will also become available for patients with SLC6A8 deficiency.
GAMT deficiency will be included in neonatal screening programs as guanidinoacetic acid seems to provide a suitable marker, as
measured in blood spots by tandem mass spectroscopy.
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Website
101. Transmembrane protein display software.
www.sacs.ucsf.edu/TOPO2
Affiliations
• Lígia S Almeida
VU University Medical Center, Department of
Clinical Chemistry, Metabolic Unit, De Boelelaan
1117, 1081 HV, Amsterdam, The Netherlands
Tel.: +31 204 442 914;
Fax: +31 204 440 305;
[email protected]
• Efraim H Rosenberg
VU University Medical Center, Department of
Clinical Chemistry, Metabolic Unit, De Boelelaan
1117, 1081 HV Amsterdam, The Netherlands
Tel.: +31 204 442 914;
Fax: +31 204 440 305;
[email protected]
Future Neurol. (2006) 1(5)
Are cerebral creatine deficiency syndromes on the radar screen? – REVIEW
• Nanda M Verhoeven
VU University Medical Center, Department of
Clinical Chemistry, Metabolic Unit,
De Boelelaan 1117, 1081 HV Amsterdam,
The Netherlands
Tel.: +31 204 442 914;
Fax: +31 204 440 305;
[email protected]
www.futuremedicine.com
• Cornelis Jakobs
VU University Medical Center, Department of
Clinical Chemistry, Metabolic Unit,
De Boelelaan 1117, 1081 HV Amsterdam,
The Netherlands
Tel.: +31 204 442 914;
Fax: +31 204 440 305;
[email protected]
• Gajja S Salomons
VU University Medical Center, Department of
Clinical Chemistry, Metabolic Unit,
De Boelelaan 1117, 1081 HV Amsterdam,
The Netherlands
Tel.: +31 204 442 914;
Fax: +31 204 440 305;
[email protected]
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